Role of Dorsal Raphe Nucleus and Ventral Tegmental Area on Reward and Feeding Behaviors in Mice

Neuroscienze e Disturbi del Comportamento Biomedicina e Neuroscienze Settore Scientifico Disciplinare BIO/14 ROLE OF DORSAL RAPHE NUCLEUS AND VENTRAL TEGMENTAL AREA ON REWARD AND FEEDING BEHAVIORS IN MICE IL DOTTORE IL COORDINATORE ROSA ANNA MARIA MARINO GIOVANNI ZUMMO IL REFERENTE IL TUTOR CARLA CANNIZZARO EMANUELE CANNIZZARO XXVI CICLO ANNO CONSEGUIMENTO TITOLO 2016 INDEX Preface pag.3 Introduction The heterogeneity of ventral tegmental area neurons: projection functions in a moodrelated context pag.4 Morphological and Neurochemical Characterization Of Dorsal Raphe Nucleus pag.7 Dopamine Role in Reward Function pag.9 Involvement of VTA Dopamine Neurons in Feeding and Rewarding Behavior pag.12 Aims pag.14 Experimental Procedures of DNR project Animals pag.15 Optogenetic real-time place preference pag.16 Optogenetic nose-poke self-stimulation pag.17 Electrophysiology pag.17 Results Pharmacological stimulation of dopamine but not serotonin release reinforces behavior pag.18 Optogenetic stimulation of VTA dopamine but not DRN serotonin cell bodies reinforces behavior pag.20 Optogenetic stimulation of DRN cell bodies reinforces behavior in a dopamine- dependent, serotonin-independent manner pag.22 Optogenetic stimulation of dopaminergic or GABAergic DRN cell bodies fails to reinforce nose-poke self-stimulation pag.24 1 Unlike serotonergic neurons, non-serotonergic DRN neurons preferentially project to the VTA pag.26 The majority of DRN cell bodies that project to VTA are non-serotonergic pag.28 DRN-VTA projections reinforce behavior and provide synaptic glutamatergic excitation of VTA dopamine neurons primarily via non-serotonergic projections pag.30 Discussion pag.32 Materials and methods of VTA project on feeding and reward pag.38 References pag.46 2 PREFACE Before presenting the work performed during my PhD program my main aim is to thankthe people that believed in me since I started my experience in the Neuroscience field. Firstly, wish to thank my family, my mother and my brother because they followed me during this path, too hard to take on in some moments in which I really thought to leave everything for coming back to my old, boring and easier life. I couldn’t avoid to say thank you to my teacher Carla Cannizzaro because she is the first person that introduced me the neuroscience world, sharing with me her passion for the research. In the last two years of my PhD program, I performed my research in NIDA laboratories, under the supervision of the Dr. Antonello Bonci. Even if my previous work dealt with the role of stress reaction in alcohol addiction, when I moved in NIDA I spent about four months in learning optogenetic, anatomical and immunohistochemistry tools that, coupled with my already assessed behavioral knowledge, could permit me to perform an independent experimental research. In order to do that I supported a Senior Post Doc, Ross McDevitt, who, teaching me all these techniques, involved me in his project. This project coped with the characterization of Dorsal Raphe cell types responsible for reward processes. Even if we already published a paper on September 25, 2014” Serotonergic versus non- serotonergic dorsal raphe projection neurons: differential participation in reward circuitry” we are still working on it. I was also involved in another project on Parkinson’s disease that copes with VGLUT2 role in the development of this pathology. In the last two months, looking forward my new path as post-doc in Bonci’s Lab, I launched a new project on VTA role on feeding and rewarding behaviors. 3 THE HETEROGENEITY OF VENTRAL TEGMENTAL AREA NEURONS: PROJECTION FUNCTIONS IN A MOOD-RELATED CONTEXT Dopaminergic neurons of the ventral tegmental area (VTA) play a central role in reward learning (Wise, 2004). Midbrain dopamine neurons located in the VTA play also a key role in several disorders including schizophrenia, drug addiction and mood disorders such as depression (Marinelli and White, 2000, Krishnan et al., 2007, Cao et al., 2010, Valenti et al., 2011, Chaudhury et al., 2013, Friedman et al., 2014). Even if classically the VTA was thought to consist of dopamine (DA) neurons (Yim and Mogenson, 1980, Grace and Onn, 1989), studies have shown that while the majority of cells in the VTA are dopaminergic (~70%), there are also small percentages of both GABA (~30%) and glutamatergic (~2–3%) neurons in this region (Yamaguchi et al., 2007, Nair-Roberts et al., 2008). Additionally, certain subpopulations of neurons have been shown to co-release two transmitters (Sulzer et al., 1998,Stuber et al., 2010, Tritsch et al., 2012). The advent of optogenetics has allowed for the dissection of neural circuits in both a cell-type and projection-specific manner (Lobo et al., 2010, Lammel et al., 2011, Chaudhury et al., 2013, Tye et al., 2013). Further studies in non-human primates suggested that phasic activation of DA neurons was found to serve more in denoting the occurrence in reward related-stimuli than actually mediating the hedonic effects of reward (Schultz, 1998b). More specifically, recordings in non-human primates performing an operant task demonstrated that DA neurons could be activated by conditioned, reward predicting stimuli (Schultz, 1998a). Occurrence of reward in the absence of a conditioned stimulus (CS) induces phasic activation of DA neurons. Further, it was seen that when a CS predicted the occurrence of reward phasic firing was elicited immediately following the CS prior to the onset of the reward. Finally, phasic activation of DA neurons occurs following a CS, however, in the failure of a reward, DA neurons are depressed at texact 4 expected time of the reward. Initially, many in vitro slice recording experiments, performed both in mice and rats, suggested that VTA DA neurons were a homogenous population (Ungless et al., 2001, Argilli et al., 2008, Chen et al., 2008,Stuber et al., 2008). Early in vitro electrophysiological studies, performed in Sprague-Dawley rats, classified DA neurons of the VTA as the primary population of neurons (Grace and Onn, 1989, Schultz, 1998a). However, later studies note that other populations of cells also exist within the VTA, GABAergic, as well as glutamatergic neurons (Nair-Roberts et al., 2008). The neurochemical identities of all of these neurons still remain uncharacterized. GABAergic neurons within the VTA of Sprague-Dawley rats, exhibit a large amount of heterogeneity with a large range of action potential durations and firing rates. (Margolis et al., 2012). They constitute approximately 15–20% of the entire neuronal population (Margolis et al., 2012) and synapse onto both DA and non-DA VTA neurons (Bayer and Pickel, 1991,Omelchenko and Sesack, 2009). Similar to DA VTA neurons, GABAergic VTA neurons may also play diverse roles in behavioral responses. Some neurons in the VTA of both Sprague-Dawley rats and VGLUT1 kockout mice, express vesicular glutamate transporter 2 (VGLUT2), a marker of glutamatergic neurons, and are 2–3% of the total neuronal population, being located primarily in the rostro-medial portion of the VTA (Fremeau et al., 2004, Nair-Roberts et al., 2008). All cells contain glutamate for their role in protein synthesis, however, for exocytotic release, the VGLUTs are required (Reimer and Edwards, 2004, Takamori, 2006). The VTA projects to many regions including the NAc, mPFC, and the amygdala (Wise and Bozarth, 1985). 5 While it has been established that the VTA-to-NAc circuit is a crucial element in the pathogenesis of stress-related disorders, other areas, such as the mPFC and amygdala are also known to affect these behaviors. Notably, the mPFC both receives innervations from the VTA and sends projections to the VTA and NAc, forming a regulatory feedback mechanism (Nestler and Carlezon, 2006). 6 MORPHOLOGICAL AND NEUROCHEMICAL CHARACTERIZATION OF DORSAL RAPHE NUCLEUS Whole-brain mapping studies have found the greatest density of VTA-projecting neurons to reside in the dorsal raphe nucleus (DRN) (Phillipson, 1979; Watabe-Uchida et al., 2012). The DRN contains the largest group of serotonin neurons in the brain, and supplies the vast majority of ascending serotonergic projections (Jacobs and Azmitia, 1992). The primary synaptic inputs within and to the raphe are glutamatergic and GABAergic. The DRN is divided into three subfields, i.e., ventromedial (vmDR), lateral wings (lwDR) and dorsomedial (dmDR). Although 5-HT neurons have similar physiological properties, important differences exist between subfields. Non-5-HT neurons are indistinguishable from 5-HT neurons. Glutamate neurons, as defined by vGlut3 anti-bodies, are intermixed and co-localized with 5-HT neurons within all raphe subfields. Finally, the dendritic arbor of the 5-HT neurons is distinct between subfields. Previous studies regard 5-HT neurons as a homogenous population. Understanding the interaction of the cell properties of the neurons in concert with their morphology, local distribution of GABA and glutamate neurons and their synaptic input, reveals a more complicated and heterogeneous raphe. These findings leave an open question: how specific subfields can modulate behavior? The role of the DRN in reinforcement learning is unclear, with literature suggesting both excitatory and inhibitory functions. For example, electrical stimulation of the DRN is sufficient to vigorously reinforce instrumental behavior in rats (Corbett and Wise, 1979; Margules, 1969; Rompre and Miliaressis, 1985; Simon et al., 1976; Van Der Kooy et al., 1978). In contrast, drugs that selectively elevate

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